title: FHA-2 treated PBMC, 2Hrs (Experiment Micro#22, sample #15)

description:
Simple annotation: Bordetella exposure, Filamentous Hemaglutinin (FHA)
Image: http://smd.stanford.edu/MicroArray/gifs/2003-05/41471.gif
Infection: yes
Time: 2

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 920, 5800; Parameter Datafile type = GenePix Results 1.4; Software: type: feature extraction; Performer: Christine,,Dieterich Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 33.32; Parameter Scan Time = 15:49:24; Parameter PMT Volts = 780; Parameter Scan Date = 2003-04-29; Parameter Pixel Size = 10; Parameter Laser on time = 16132; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [81969]; Parameter Laser Power = 3.60; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Christine,,Dieterich Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 33.32; Parameter Scan Time = 15:49:24; Parameter PMT Volts = 900; Parameter Scan Date = 2003-04-29; Parameter Pixel Size = 10; Parameter Laser on time = 16131; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [81969]; Parameter Laser Power = 3.58; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Christine,,Dieterich


title: Mock-1 treated PBMC, time point 2Hrs (Experiment Micro#22, sample #12)

status: Public on Oct 30 2008

type: RNA

city: Palo Alto

first: Christine

last: Dieterich

state: CA

address: 3801 Miranda Avenue

department: Department of Microbiology & Immunology

country: USA

institute: VA hospital

description:
Simple annotation: Bordetella exposure, Controls
Image: http://smd.stanford.edu/MicroArray/gifs/2003-05/41468.gif
Infection: mock-treatment
Time: 2

hyb_protocol: not provided

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 920, 5800; Parameter Datafile type = GenePix Results 1.4; Software: type: feature extraction; Performer: Christine,,Dieterich Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 33.07; Parameter Scan Time = 15:09:44; Parameter PMT Volts = 790; Parameter Scan Date = 2003-04-29; Parameter Pixel Size = 10; Parameter Laser on time = 16093; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [81969]; Parameter Laser Power = 3.60; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Christine,,Dieterich Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 33.07; Parameter Scan Time = 15:09:44; Parameter PMT Volts = 880; Parameter Scan Date = 2003-04-29; Parameter Pixel Size = 10; Parameter Laser on time = 16092; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [81969]; Parameter Laser Power = 3.59; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Christine,,Dieterich

data_processing: VALUE is Log (base 2) of the ratio of the median of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm)

molecule: total RNA

organism: Homo sapiens

source_name: Stratagene Universal Human Reference  RNA amplified 1x using Ambion MessageAmp aRNA kit

label_protocol: not provided

extract_protocol: not provided

source_name: 1 ug total RNA from 4x10E6 Mock-1 treated cells , amplified 1x using Ambion MessageAmp aRNA kit

treatment_protocol: Dieterich_Treatment of human PBMC with purified Bordetella pertussis filamentous hemagglutinin (FHA); Preparation of human peripheral blood mononuclear cells (PBMC)and Bordetella pertussis filamentous hemagglutinin (FHA):Human PBMCs were purified from freshly drawn blood or buffy coat from healthy donors (Stanford Blood Center, Stanford,  CA), using Ficoll-Paque (Amersham Biosciences, Piscataway, N.J.) according to the manufacturer?s instruction. PBMCs were resuspended in RPMI-1640 medium (American Type Culture Collection, ATCC, Manassas, VA) supplemented with 10% fetal bovine serum (ATCC), 100 U/ml penicillin, and 100 ug/ml streptomycin (Gibco, Invitrogen Corportation, Carlsbad, CA). FHA was purified from B. pertussis Bpe160, Bpe162, and Bpe163 and named FHA-2, FHA-3, and FHA-4, respectively, in accordance with a previously described protocol. Briefly, B. pertussis liquid cultures were harvested when the optical density at 600 nm was between 2.5 and 4. The culture supernatant was supplemented with Complete Protease Inhibitor (Roche, Indianapolis, IN) and applied at 2 ml/min on a 5 ml HiTrap Heparin column (Amersham Biosiciences). Purification was performed using an FPLC system equipped with a P-500 pump and a FRAC-P100 fraction collector (Pharmacia LKB). FHA was eluted at 2 ml/min with 1X PBS, 0.5 M NaCl (Mock-2). Total protein concentrations, determined by the BCA test (Pierce, Rockford, IL), were 560, 300, and 340 ug/ml for FHA-2, FHA-3, and FHA-4, respectively. FHA-1 (a kind gift from M. Pizza and R. Rappuoli, Chiron, Sienna, Italy) was isolated from a derivative of B. pertussis, Wellcome 28 (W28) using Matrex Cellufine Sulfate (Millipore) and stored in a solution containing 50% glycerol, 0.5 M NaCl, and 25 mM Na2HPO4 (Mock-1).  PBMC treatment: Cultures comprising 3x106 PBMC were stimulated for 0.5, 2, 4, and 6 hours with 5 ug/ml of each purified B. pertussis FHA. Control PBMC cultures were simultaneously treated with similar volumes of elution buffers (Mock-1 and Mock-2), and sampled at the same time points.; Protocol Type = Biological Sample; Performer: Christine,,Dieterich


title: Bovine 4 Cell, biological rep2

status: Public on Oct 27 2008

city: Berlin

first: Smita

last: Sudheer

email: sudheer@molgen.mpg.de, adjaye@molgen.mpg.de, kues@tzv.fal.de, niemann@tzv.fal.de

address: Ihnestr. 63-73

country: Germany

institute: Max Planck Institute for Molecular Genetics

description: Gene expression data from preimplantation embryos 4 Cell

hyb_protocol: Ten μg of fragmented cRNA was used for hybridization on the GenChip Bovine Genome Array (Affymetrix) overnight at 45°C according to the manufacture’s instructions.

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray Scanner.

data_processing: The Bioconductor open source library was used for initial processing of the raw files. CEL files were preprocessed using the GCRMA algorithm of the GCRMA package [v2.0.0] and detection calls were obtained using the mas5calls function of the Affymetrix platform.

label: biotin

molecule: polyA RNA

organism: Bos taurus

source_name: Bovine preimplantation embryo Invivo derived 4 Cell

label_protocol: biotin-labeled cRNA was prepared in an in vitro transcription reaction using the GeneChip IVT Labeling Kit (Affymetrix).

extract_protocol: Poly(A)+ RNA was isolated with a Dynabeads mRNA Direct Kit (Invitrogen) according to the manufacturer's instructions.

tag: In vivo derived

value: 4 Cell


title: Bovine 8 Cell, biological rep3

description: Gene expression data from preimplantation embryos 8 Cell

source_name: Bovine preimplantation embryo Invivo derived 8 Cell

value: 8 Cell


title: Untreated PBMC, timem point 0 Hr (Experiment Micro#22, sample #4)

description:
Simple annotation: Bordetella exposure, untreated
Image: http://smd.stanford.edu/MicroArray/gifs/2003-05/41460.gif
Infection: no
Time: 0

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 920, 5800; Parameter Datafile type = GenePix Results 1.4; Software: type: feature extraction; Performer: Christine,,Dieterich Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 31.55; Parameter Scan Time = 12:27:35; Parameter PMT Volts = 810; Parameter Scan Date = 2003-04-29; Parameter Pixel Size = 10; Parameter Laser on time = 15932; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [81969]; Parameter Laser Power = 3.61; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Christine,,Dieterich Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 31.55; Parameter Scan Time = 12:27:35; Parameter PMT Volts = 910; Parameter Scan Date = 2003-04-29; Parameter Pixel Size = 10; Parameter Laser on time = 15931; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [81969]; Parameter Laser Power = 3.53; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Christine,,Dieterich

tag: Stratagene Universal Human Reference  RNA amplified 1x using Ambion MessageAmp aRNA kit

source_name: 1 ug total RNA from 4x10E6 untreated cells , amplified 1x using Ambion MessageAmp aRNA kit


title: Bovine 8 Cell, biological rep1


title: Bovine 8 Cell, biological rep2


title: Mock-2 treated PBMC, time point 0.5Hr (Experiment Micro#22, sample #8)

description:
Simple annotation: Bordetella exposure, Controls
Image: http://smd.stanford.edu/MicroArray/gifs/2003-05/41465.gif
Infection: mock-treatment
Time: 0.5

scan_protocol: Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 920, 5800; Parameter Datafile type = GenePix Results 1.4; Software: type: feature extraction; Performer: Christine,,Dieterich Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 32.55; Parameter Scan Time = 13:32:13; Parameter PMT Volts = 800; Parameter Scan Date = 2003-04-29; Parameter Pixel Size = 10; Parameter Laser on time = 15996; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [81969]; Parameter Laser Power = 3.61; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Christine,,Dieterich Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 32.55; Parameter Scan Time = 13:32:13; Parameter PMT Volts = 900; Parameter Scan Date = 2003-04-29; Parameter Pixel Size = 10; Parameter Laser on time = 15995; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000B [81969]; Parameter Laser Power = 3.55; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Christine,,Dieterich

source_name: 1 ug total RNA from 4x10E6 Mock-2 treated cells , amplified 1x using Ambion MessageAmp aRNA kit


title: Bovine 4 Cell, biological rep1


title: Bovine 2 Cell, biological rep2

description: Gene expression data from preimplantation embryos 2 Cell

source_name: Bovine preimplantation embryo Invivo derived 2 Cell

value: 2 Cell


title: Bovine Zygote, biological rep1

description: Gene expression data from preimplantation embryos Zygote

source_name: Bovine Invivo derived Zygotes

value: Zygote


title: Bovine Oocyte invivo, biological rep3

description: Gene expression data from  Invivo cultured Oocyte

source_name: Bovine Invivo cultured Oocytes

value: Oocyte


title: Bovine Oocyte invivo, biological rep1


title: Bovine Oocyte invitro, biological rep2

description: Gene expression data from  Invitro cultured Oocyte

source_name: Bovine Invitro cultured Oocytes

tag: In vitro cultured


title: Bovine Morula, biological rep2

description: Gene expression data from preimplantation embryos Morula

source_name: Bovine preimplantation embryo Invivo derived Morula

value: Morula


title: Bovine 2 Cell, biological rep1


title: Bovine Oocyte invivo, biological rep2


title: Bovine Zygote, biological rep3


title: Bovine Oocyte invitro, biological rep1


title: Bovine Oocyte invitro, biological rep3


title: Bovine Zygote, biological rep2


title: WCE in HU

status: Public on Oct 10 2008

type: genomic

city: Bunkyo-ku

first: Katsuhiko

last: Shirahige

state: Tokyo

address: 1-1-1 Yayoi

department: Research Center for Epigenetic Disease

country: Japan

institute: The University of Tokyo

description: WCE sample used for normalization of Pol2 Chip Data.

hyb_protocol: Hybridization, blocking and washing steps were carried out as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). Each sample was hybridized to the array in 150ul containing 6XSSPE, 0.005% TritonX-100, 15ug fragmented denatured salmon sperm DNA (Gibco-BRL), and 1nmole of a 3’-biotin control oligonucleotide (oligo B2 probes) that hybridized to the border features on the array. Samples were heated to 100°C for 10 min., and then cooled on ice. Samples were hybridized for 16h at 42°C in a hybridization oven (GeneChip hybri oven 320, Affymetrix, CA). Washing and staining protocol (Mini_euk1 ver2) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix, CA).

scan_protocol: Each array was scanned by HP GeneArray Scanner (Affymetrix, CA) at an emission wavelength of 560nm at 7.5uM resolution. Grids were aligned to the scanned images using the know feature of the array. Primary data analyses were carried out using the Affymetrix Microarray Suite Ver.5.0 software to obtain hybridization intensity, fold change value, change p-value, and detection p-value for each locus.

data_processing: For the discrimination of positive and negative signals for the binding, we compared ChIP fraction with Sup (supernatant) fraction by using three criteria as follows. First, the reliability of strength of signal was judged by detection p-value of each locus (p-value lower than 0.025 was considered as significant). Secondly, reliability of binding ratio was judged by change p-value (p-value lower than 0.025 was considered as significant). Thirdly, clusters consisted of at least three contiguous loci which filled above two criteria were selected as significantly enriched locus, because it is apparent that a single site of protein-DNA interaction will result in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. This third criterion is very unique, make the data highly reliable and can be only applicable to the chip data obtained by our high-resolution tiling array.

molecule: genomic DNA

organism: Saccharomyces cerevisiae

source_name: ChIP DNA

label_protocol: DNA was end-labelled with Biotin-N6ddATP by Terminal Transferase as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998)

extract_protocol: 1.5X10^8 cells were disrupted by MULTI-BEADS SHOCKER (MB400U, YASUI KIKAI, Osaka), which was able to keep cells precisely at lower than 6°C during disruption by glass beads. This enabled us to retrieve more than 60% of tagged proteins to soluble fraction routinely. Chromosomal DNA was shared into the average size of 400-600bp by sonication. Anti-HA monoclonal antibody HA.11 (16B12) (CRP Inc., Denver, PA) and anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) were used for chromatin immuno-precipitation. Immunoprecipitated chromosomal DNA was subsequently purified following the protocol of Young’s lab (Genome wide location analysis web page, MIT). Purified DNA was random primed and amplified by the protocol of Brown’s lab (Stanford). 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998).

tag: WCE from HU arrested Cells.


title: DNA Pol2 distribution in HU

description:
DNA Pol2 binding profile after 1h in HU.
For normalization of this sample WCE (GSM305882) was used.

tag: Pol2-3XHA was immuno-precipitated from HU arrested Wild Type Cells.


title: DNA Pol2 distribution in tof1 deleted in HU

tag: Pol2-3XHA was immuno-precipitated from HU arrested in tof1 deleted Cells.


title: DNA Pol2 distribution in mrc1 deletion mutant

source_name: DNA Pol2-3XHA ChIP DNA

tag: Pol2-3XHA was immuno-precipitated from HU arrested mrc1 deleted cells.


title: 13742592

status: Public on Oct 15 2008

city: Bozeman

first: Robert

middle: A.

last: Cramer

state: MT

department: Department of Veterinary Molecular Biology

institute: Montana State University

description: Aspergillus

hyb_protocol: hybridization and slide processed according to TIGR/JCVI protocol

scan_protocol: scanning following Axon instruments 4000B scanner instruction manual using GenePix software

data_processing: MIDAS normalizing using LOWESS, Standard Deviation Regularization and In-slide replica analysis

organism: Aspergillus fumigatus

source_name: synthetic_DNA from wild-type strain CEA10 labeled with Cy3

label_protocol: cDNA synthesis and aa-labeling following instruction of the TIGR protocols

growth_protocol: strains were grown in liquid cultures for 24h under normoxic conditions then shifted for 24h to hypoxic conditions

extract_protocol: RNA extraction with TRIzol and Qiagen RNeasy Mini Kit columns after manufactor instructions

treatment_protocol: mycelia were harvested by drying on a Buchner funnel and then snap frozen at -80C. Prior to RNA extraction frozen pellets were homogenized by a beadbeater

tag: strain CEA10, 24hr hypoxic conditions

source_name: synthetic_DNA from srbA mutant labeled with Cy5

tag: strain srbA mutant, 24hr hypoxic conditions


title: Ex-smoker, Squamous cell lung carcinoma T35 (smoke)

status: Public on Oct 01 2008

city: Groningen

first: Mirjam

last: Boelens

address: Hanzeplein 1

country: Netherlands

institute: UMCG

hyb_protocol: Hybridization was performed with 750 ng of each labeled target together with fragmentation- and hybridization buffer at 60C for 17 hours onto Agilent Whole Human Genome oligoarrays following manufacturer’s protocol.

scan_protocol: The microarray slides were washed following the user manual instructions and scanned using the Agilent dual laser DNA microarray scanner. The microarray data were normalized using the Agilent feature extraction software v. 8.5 (Agilent Technologies, Palo Alto, CA) with regards to no background correction.

data_processing: Quantile normalization was applied to log2 transformed median signals of Cy5 and Cy3 intensities. Principal component analysis was performed and samples with a factor loading with a first principal component of more than two times the standard deviation from the mean were excluded as their hybridisations were considered to be of low quality. Intensities were standardised by subtracting the variance explained by the first principal component, as this is gene specific variation that does not contribute to differences between arrays. Probe sequences that were spotted more than once were averaged. Datasets of Cy3 and Cy5 were averaged.

label: Cy3 and Cy5

source_name: T35, ex-smoker

label_protocol:
ULS LABELING
1. Take 1- 2 μg of aRNA. Ensure that the final concentration in the labeling reaction is above 50 ng/μL. Optimal modification degrees of the labeled material are not achieved if the final concentration of aRNA in the labeling mixture is below 50 ng/μL)
2. Add 1 μL of Cy5-ULS or Cy3-ULS per 1 μg aRNA (Always keep the ratio of μg of aRNA to μL ULS 1:1 when increasing or decreasing the amount to be labeled).
3. Add 1/10 volume of 10 x Labeling solution
4. Adjust with RNase-free water to final volume and mix by pipetting
5. Label sample by incubation for 15 minutes at 85°C
6. Place samples on ice, spin down to collect contents of tube before proceeding with purification using the KREApure columns
Dye Removal using KREApure Columns (Removal of free ULS label using KREApure columns
(20800 x g is equivalent to 14,000 rpm on eppendorf 5417C)):
1. Resuspend column material by vortexing
2. Loosen cap ¼ turn and snap off the bottom closure
3. Place the column in a 2 mL collection tube
4. Pre-spin the column for 1 minute at 20800 x g
5. Discard flow through and column cap, but re-use collection tube
6. Add 300 μL ultrapure water to the column and centrifuge for 1 min at 20,800g
7. Discard collection tube and flow-through
8. Place column in a new RNase free 1.5 mL microcentrifuge tube (not provided)
9. Add ULS-labeled aRNA onto column bed
10. Centrifuge column for 1 minute at 20800 x g
11. Flow through contains the purified labeled aRNA

extract_protocol:
LASER DISSECTION MICROSCOPY
- Frozen squamous cell lung carcinoma tissue sections of 8μm were used for laser dissection microscopy (LDM)
- Only vital tumor cells without apparent admixture of inflammatory cells through the tumor fields were isolated using laser dissection microscopy
- An area of approximately 20x106 μm2 was microdissected from the section using P.A.L.M. Microlaser Technology system (P.A.L.M., Bernried, Germany).
- Laser microdissected cells were collected in lysis buffer (Macherey-Nagel)
RNA ISOLATION
Total RNA was isolated and purified from the microdissected cells with a Nucleospin RNA II kit (Macherey-Nagel). The quantity of DNA-free total RNA was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA quality was assessed by the presence and ratio of 18S and 28S rRNA bands combined with a low baseline, monitored with RNA 6000 PicoChip (Agilent, Palo Alto, CA) on the 2100 bioanalyzer (Agilent).
RNA AMPLIFICATION
For each sample, 150 ng total RNA was amplified using the MessageAmp II kit (Ambion, Austin, USA). Briefly, 150 ng total RNA was mixed with 1 l of T7 Oligo(dT) primer in a total volume of 12 l. Primer and template were denatured by incubating at 70C for 10 minutes and annealed by putting the reaction tubes on ice. The First Strand Reaction was performed by adding 8 µl Reverse Transcription Master Mix (containing 10x First Strand buffer, Ribonuclease Inhibitor, dNTP Mix and Reverse Transcriptase) and incubating at 42C for 2 hours. Second Strand cDNA Synthesis was done by adding 63 µl Nuclease-Free Water, 10 µl 10x Second Strand Buffer, 4µl dNTP Mix, 2µl DNA Polymerase and 1 µl RNase H and incubating at 16C for 2 hours. cDNA purification was done according manufacturers protocol (Ambion). In vitro transcription was initiated by addition of 2 µl aaUTP Solution (50mM), 12 µl ATP, CTP, GTP mix (25mM), 3 µl UTP Solution (50mM), 4 µl T7 10x Reaction Buffer and 4 µl T7 Enzyme Mix and incubated at 37C for 9 hours. Machery-Nagel RNA Clean up mini spin columns are used for purification of the cRNA.

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

value: male

tag: Age at time of surgery:	62

tag: Smoking status:		ex

tag: Packyears:			25

tag: Type of Surgery:		pneumectomy

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		left upper lobe

tag: TNM stage:			T3N1M0

tag: Tumor stage:			IIIA

tag: Dead/alive at last follow-up:	alive

tag: Survival as from surgery:	98 months

tag: Metastases:			lymph node

tag: Time to distant metastasis:	not applicable

tag: COPD:				COPD

tag: FEV1/FVC:			66 %

tag: GOLD stage:			GOLD II


title: 13742591

description: Aspergillus

tag: strain CEA10, 24hr hypoxic conditions

tag: strain srbA mutant, 24hr hypoxic conditions


title: 13742589

description: Aspergillus

source_name: synthetic_DNA from srbA mutant labeled with Cy3

source_name: synthetic_DNA from wild-type strain CEA10 labeled with Cy5


title: Current-smoker, Squamous cell lung carcinoma T31 (smoke)

source_name: T31, current smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

value: female

tag: Age at time of surgery:	61

tag: Smoking status:		current

tag: Packyears:			35

tag: Location tumor:		right upper lobe

tag: TNM stage:			T2N1M0

tag: Tumor stage:			IIB

tag: Survival as from surgery:	85 months

tag: FEV1/FVC:			65 %

tag: GOLD stage:			GOLD I


title: Ex-smoker, Squamous cell lung carcinoma T28 (smoke)

source_name: T28, ex-smoker

tag: Age at time of surgery:	76

tag: Smoking status:		ex

tag: Packyears:			40

tag: Type of Surgery:		pneumectomy

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		left upper lobe

tag: TNM stage:			T3N1M0

tag: Tumor stage:			IIIA

tag: Dead/alive at last follow-up:	dead

tag: Survival as from surgery:	58 months

tag: Metastases:			lymph node

tag: Time to distant metastasis:	not applicable

tag: COPD:				no COPD

tag: FEV1/FVC:			73 %

tag: GOLD stage:			not applicable


title: Ex-smoker, Squamous cell lung carcinoma T32 (smoke)

source_name: T32, ex-smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	63

tag: Location tumor:		left lower lobe

tag: TNM stage:			T2N1M0

tag: Tumor stage:			IIB

tag: Survival as from surgery:	91 months

tag: COPD:				COPD

tag: FEV1/FVC:			62 %


title: Current-smoker, Squamous cell lung carcinoma T33 (smoke)

source_name: T33, current smoker

tag: Age at time of surgery:	51

tag: Smoking status:		current

tag: Packyears:			33

tag: Type of Surgery:		pneumectomy

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		right middle lobe

tag: Dead/alive at last follow-up:	alive

tag: Survival as from surgery:	69 months

tag: FEV1/FVC:			64 %

tag: GOLD stage:			GOLD I


title: unknown-smoker, Squamous cell lung carcinoma T29 (smoke)

data_processing: Quantile normalization was applied to log2 transformed median signals of Cy5 and Cy3 intensities. Principal component analysis was performed and samples with a factor loading with a first principal component of more than two times the standard deviation from the mean were excluded as their hybridisations were considered to be of low quality. For this sample T29, dataset of Cy3 was excluded due to low quality. Intensities were standardised by subtracting the variance explained by the first principal component, as this is gene specific variation that does not contribute to differences between arrays. Probe sequences that were spotted more than once were averaged.

source_name: T29, unknown smoking status

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	58

tag: Smoking status:		unknown

tag: Packyears:			10

tag: Location tumor:		right lower lobe

tag: TNM stage:			T2N2M0

tag: Tumor stage:			IIIA

tag: Survival as from surgery:	130 months

tag: Metastases:			lymph node

tag: Time to distant metastasis:	not applicable

tag: COPD:				no COPD

tag: FEV1/FVC:			71 %

tag: GOLD stage:			not applicable


title: Current-smoker, Squamous cell lung carcinoma T23 (smoke)

source_name: T23, current smoker

tag: Age at time of surgery:	60

tag: Smoking status:		current

tag: Packyears:			40

tag: Type of Surgery:		lobectomy

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		right upper lobe

tag: TNM stage:			T3N0M0

tag: Tumor stage:			IIB

tag: Dead/alive at last follow-up:	dead

tag: Survival as from surgery:	9 months

tag: Metastases:			distant

tag: Time to distant metastasis:	6 months

tag: FEV1/FVC:			73 %


title: Current-smoker, Squamous cell lung carcinoma T24 (smoke)

source_name: T24, current smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	67

tag: Packyears:			45

tag: Type of Surgery:		pneumectomy

tag: Location tumor:		right lower lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Survival as from surgery:	16 months

tag: Time to distant metastasis:	16 months

tag: COPD:				COPD

tag: FEV1/FVC:			63 %

tag: GOLD stage:			GOLD I


title: 13742588

description: Aspergillus

tag: strain srbA mutant, 24hr hypoxic conditions

tag: strain CEA10, 24hr hypoxic conditions


title: Current-smoker, Squamous cell lung carcinoma T25 (smoke)

source_name: T25, current smoker

tag: Age at time of surgery:	66

tag: Smoking status:		current

tag: Packyears:			50

tag: Type of Surgery:		lobectomy

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		left upper lobe

tag: TNM stage:			T3N0M0

tag: Tumor stage:			IIB

tag: Dead/alive at last follow-up:	alive

tag: Survival as from surgery:	20 months

tag: Metastases:			distant

tag: Time to distant metastasis:	11 months

tag: FEV1/FVC:			58 %

tag: GOLD stage:			GOLD II


title: Ex-smoker, Squamous cell lung carcinoma T34 (smoke)

source_name: T34, ex-smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	54

tag: Smoking status:		ex

tag: Packyears:			30

tag: Type of Surgery:		pneumectomy

tag: Location tumor:		right lower lobe

tag: TNM stage:			T3N1M0

tag: Tumor stage:			IIIA

tag: Survival as from surgery:	128 months

tag: Metastases:			lymph node

tag: Time to distant metastasis:	not applicable

tag: COPD:				COPD

tag: FEV1/FVC:			61 %


title: Ex-smoker, Squamous cell lung carcinoma T21 (smoke)

source_name: T21, ex-smoker

tag: Age at time of surgery:	69

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		right middle lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Dead/alive at last follow-up:	dead

tag: Survival as from surgery:	6 months

tag: Metastases:			distant

tag: Time to distant metastasis:	4 months

tag: COPD:				no COPD

tag: FEV1/FVC:			76 %

tag: GOLD stage:			not applicable


title: Ex-smoker, Squamous cell lung carcinoma T30 (smoke)

source_name: T30, ex-smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	71

tag: Smoking status:		ex

tag: Packyears:			3

tag: Type of Surgery:		pneumectomy

tag: Location tumor:		right lower lobe

tag: TNM stage:			T2N1M0

tag: Tumor stage:			IIB

tag: Survival as from surgery:	71 months

tag: FEV1/FVC:			72 %


title: Current-smoker, Squamous cell lung carcinoma T15 (smoke)

source_name: T15, current smoker

tag: Age at time of surgery:	47

tag: Smoking status:		current

tag: Packyears:			30

tag: Type of Surgery:		lobectomy

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		left lower lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Dead/alive at last follow-up:	alive

tag: Survival as from surgery:	72 months

tag: Metastases:			no

tag: Time to distant metastasis:	not applicable

tag: COPD:				COPD

tag: FEV1/FVC:			53 %

tag: GOLD stage:			GOLD II


title: Current-smoker, Squamous cell lung carcinoma T16 (smoke)

source_name: T16, current smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	74

tag: Packyears:			39

tag: Type of Surgery:		bilobectomy

tag: Location tumor:		right upper lobe

tag: Dead/alive at last follow-up:	dead

tag: Survival as from surgery:	75 months

tag: FEV1/FVC:			49 %


title: Current-smoker, Squamous cell lung carcinoma T27 (smoke)

source_name: T27, current smoker

tag: Age at time of surgery:	75

tag: Smoking status:		current

tag: Packyears:			40

tag: Type of Surgery:		lobectomy

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		left upper lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Survival as from surgery:	6 months

tag: Metastases:			distant

tag: Time to distant metastasis:	2 months

tag: FEV1/FVC:			66 %


title: Current-smoker, Squamous cell lung carcinoma T20 (smoke)

source_name: T20, current smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	71

tag: Packyears:			57

tag: Survival as from surgery:	28 months

tag: Time to distant metastasis:	28 months

tag: COPD:				no COPD

tag: FEV1/FVC:			75 %

tag: GOLD stage:			not applicable


title: Current-smoker, Squamous cell lung carcinoma T26 (smoke)

data_processing: Quantile normalization was applied to log2 transformed median signals of Cy5 and Cy3 intensities. Principal component analysis was performed and samples with a factor loading with a first principal component of more than two times the standard deviation from the mean were excluded as their hybridisations were considered to be of low quality. Intensities were standardised by subtracting the variance explained by the first principal component, as this is gene specific variation that does not contribute to differences between arrays. Probe sequences that were spotted more than once were averaged. The four datasets of the duplicates and Cy3 and Cy5 were averaged, resulting in one processed value for further analysis.

source_name: T26, current smoker

tag: Age at time of surgery:	48

tag: Smoking status:		current

tag: Packyears:			25

tag: Type of Surgery:		bilobectomy

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		left lower lobe

tag: Dead/alive at last follow-up:	dead

tag: Survival as from surgery:	34 months

tag: Time to distant metastasis:	19 months

tag: COPD:				COPD

tag: FEV1/FVC:			54 %

tag: GOLD stage:			GOLD II


title: Ex-smoker, Squamous cell lung carcinoma T11 (smoke)

source_name: T11, ex-smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	76

tag: Smoking status:		ex

tag: Packyears:			10

tag: Type of Surgery:		pneumectomy

tag: Location tumor:		left upper lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Survival as from surgery:	105 months

tag: Metastases:			no

tag: Time to distant metastasis:	not applicable

tag: GOLD stage:			GOLD I


title: Current-smoker, Squamous cell lung carcinoma T18 (smoke)

source_name: T18, current smoker

tag: Age at time of surgery:	70

tag: Smoking status:		current

tag: Packyears:			36

tag: Type of Surgery:		bilobectomy

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		right upper lobe

tag: Dead/alive at last follow-up:	dead

tag: Survival as from surgery:	23 months

tag: Metastases:			distant

tag: Time to distant metastasis:	9 months

tag: COPD:				COPD


title: Current-smoker, Squamous cell lung carcinoma T12 (smoke)

source_name: T12, current smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	73

tag: Packyears:			35

tag: Type of Surgery:		lobectomy

tag: Location tumor:		left upper lobe

tag: TNM stage:			T2N0M0

tag: Dead/alive at last follow-up:	alive

tag: Survival as from surgery:	92 months

tag: FEV1/FVC:			68 %


title: Ex-smoker, Squamous cell lung carcinoma T10 (smoke)

source_name: T10, ex-smoker

tag: Age at time of surgery:	74

tag: Smoking status:		ex

tag: Packyears:			53

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		right lower lobe

tag: TNM stage:			T1N0M0

tag: Tumor stage:			IA

tag: Survival as from surgery:	117 months

tag: Metastases:			no

tag: Time to distant metastasis:	not applicable

tag: FEV1/FVC:			69 %

tag: GOLD stage:			GOLD I


title: Current-smoker, Squamous cell lung carcinoma T17 (smoke)

source_name: T17, current smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	72

tag: Smoking status:		current

tag: Packyears:			60

tag: Type of Surgery:		bilobectomy

tag: Location tumor:		right upper lobe

tag: TNM stage:			T3N0M0

tag: Tumor stage:			IIB

tag: Survival as from surgery:	18 months

tag: Metastases:			distant

tag: Time to distant metastasis:	6 months

tag: COPD:				no COPD

tag: FEV1/FVC:			77 %

tag: GOLD stage:			not applicable


title: Current-smoker, Squamous cell lung carcinoma T13 (smoke)

source_name: T13, current smoker

tag: Age at time of surgery:	51

tag: Packyears:			28

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		right lower lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Dead/alive at last follow-up:	alive

tag: Survival as from surgery:	106 months

tag: Metastases:			no

tag: Time to distant metastasis:	not applicable

tag: COPD:				COPD

tag: FEV1/FVC:			64 %

tag: GOLD stage:			GOLD I


title: Ex-smoker, Squamous cell lung carcinoma T07 (smoke)

source_name: T07, ex-smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	61

tag: Smoking status:		ex

tag: Packyears:			25

tag: Type of Surgery:		lobectomy

tag: Survival as from surgery:	104 months

tag: FEV1/FVC:			78 %


title: Current-smoker, Squamous cell lung carcinoma T19 (smoke)

source_name: T19, current smoker

tag: Age at time of surgery:	71

tag: Smoking status:		current

tag: Packyears:			51

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		left upper lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Dead/alive at last follow-up:	dead

tag: Survival as from surgery:	13 months

tag: Metastases:			distant

tag: Time to distant metastasis:	13 months

tag: COPD:				no COPD

tag: FEV1/FVC:			79 %

tag: GOLD stage:			not applicable


title: Ex-smoker, Squamous cell lung carcinoma T09 (smoke)

source_name: T09, ex-smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	83

tag: Smoking status:		ex

tag: Packyears:			45

tag: Type of Surgery:		lobectomy

tag: Dead/alive at last follow-up:	alive

tag: Survival as from surgery:	37 months

tag: Metastases:			no

tag: Time to distant metastasis:	not applicable

tag: COPD:				COPD

tag: FEV1/FVC:			64 %

tag: GOLD stage:			GOLD I


title: Current-smoker, Squamous cell lung carcinoma T06 (smoke)

source_name: T06, current smoker

tag: Age at time of surgery:	63

tag: Smoking status:		current

tag: Packyears:			47

tag: Type of Surgery:		pneumectomy

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		left lower lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Survival as from surgery:	61 months

tag: COPD:				no COPD

tag: FEV1/FVC:			72 %


title: Ex-smoker, Squamous cell lung carcinoma T14 (smoke)

data_processing: Quantile normalization was applied to log2 transformed median signals of Cy5 and Cy3 intensities. Principal component analysis was performed and samples with a factor loading with a first principal component of more than two times the standard deviation from the mean were excluded as their hybridisations were considered to be of low quality. Intensities were standardised by subtracting the variance explained by the first principal component, as this is gene specific variation that does not contribute to differences between arrays. Probe sequences that were spotted more than once were averaged. Datsets of duplicates and of Cy3 and Cy5 were all averaged to one single value per probe.

source_name: T14, ex-smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	72

tag: Smoking status:		ex

tag: Packyears:			53

tag: Location tumor:		left upper lobe

tag: Dead/alive at last follow-up:	alive

tag: Survival as from surgery:	84 months

tag: FEV1/FVC:			65 %

tag: GOLD stage:			GOLD II


title: Ex-smoker, Squamous cell lung carcinoma T05 (smoke)

source_name: T05, ex-smoker

tag: Age at time of surgery:	59

tag: Packyears:			6

tag: Type of Surgery:		pneumectomy

tag: Tumor type:			squamous cell lung carcinoma

tag: Survival as from surgery:	93 months

tag: Metastases:			no

tag: Time to distant metastasis:	not applicable

tag: FEV1/FVC:			77 %

tag: GOLD stage:			not applicable


title: Ex-smoker, Squamous cell lung carcinoma T04 (smoke)

source_name: T04, ex-smoker

tag: Sample code

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Gender

tag: Age at time of surgery:	76

tag: Packyears:			55

tag: Type of Surgery:		lobectomy

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Dead/alive at last follow-up:	dead

tag: Survival as from surgery:	72 months

tag: COPD:				no COPD

tag: FEV1/FVC:			70 %


title: Current-smoker, Normal B22 (smoke)

data_processing: Quantile normalization was applied to log2 transformed median signals of Cy5 and Cy3 intensities. Principal component analysis was performed and samples with a factor loading with a first principal component of more than two times the standard deviation from the mean were excluded as their hybridisations were considered to be of low quality. Intensities were standardised by subtracting the variance explained by the first principal component, as this is gene specific variation that does not contribute to differences between arrays. Probe sequences that were spotted more than once were averaged. For every sample, datasets of Cy3 and Cy5 were averaged.

source_name: B22, current smoker

extract_protocol:
LASER DISSECTION MICROSCOPY
- Frozen bronchial wall of 8μm were used for laser dissection microscopy (LDM)
- Only histologically normal bronchial epithelial cells were isolated using laser dissection microscopy
- An area of approximately 25x106 μm2 was microdissected from the section using P.A.L.M. Microlaser Technology system (P.A.L.M., Bernried, Germany).
- Laser microdissected cells were collected in lysis buffer (Macherey-Nagel)
RNA ISOLATION
Total RNA was isolated and purified from the microdissected cells with a Nucleospin RNA II kit (Macherey-Nagel). The quantity of DNA-free total RNA was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA quality was assessed by the presence and ratio of 18S and 28S rRNA bands combined with a low baseline, monitored with RNA 6000 PicoChip (Agilent, Palo Alto, CA) on the 2100 bioanalyzer (Agilent).
RNA AMPLIFICATION
For each sample, 150 ng total RNA was amplified using the MessageAmp II kit (Ambion, Austin, USA). Briefly, 150 ng total RNA was mixed with 1 l of T7 Oligo(dT) primer in a total volume of 12 l. Primer and template were denatured by incubating at 70C for 10 minutes and annealed by putting the reaction tubes on ice. The First Strand Reaction was performed by adding 8 µl Reverse Transcription Master Mix (containing 10x First Strand buffer, Ribonuclease Inhibitor, dNTP Mix and Reverse Transcriptase) and incubating at 42C for 2 hours. Second Strand cDNA Synthesis was done by adding 63 µl Nuclease-Free Water, 10 µl 10x Second Strand Buffer, 4µl dNTP Mix, 2µl DNA Polymerase and 1 µl RNase H and incubating at 16C for 2 hours. cDNA purification was done according manufacturers protocol (Ambion). In vitro transcription was initiated by addition of 2 µl aaUTP Solution (50mM), 12 µl ATP, CTP, GTP mix (25mM), 3 µl UTP Solution (50mM), 4 µl T7 10x Reaction Buffer and 4 µl T7 Enzyme Mix and incubated at 37C for 9 hours. Machery-Nagel RNA Clean up mini spin columns are used for purification of the cRNA.

tag: Cell type:			histologically normal bronchial epithelial cells that were laser microdissected from frozen bronchial wall tissue

tag: Age at time of surgery:	53

tag: Smoking status:		current

tag: Packyears:			40

tag: Surgery due to:		lung transplantation

tag: Type of Surgery:		lung-explantation

tag: Location tumor:		right upper lobe (adenocarcinoma)

tag: TNM stage:			T1N0M0

tag: COPD:				COPD

tag: FEV1/FVC:			34 %

tag: GOLD stage:			GOLD IV


title: Ex-smoker, Normal B25 (smoke)

source_name: B25, ex-smoker

tag: Sample code

tag: Gender

tag: Age at time of surgery:	59

tag: Smoking status:		ex

tag: Packyears:			29

tag: Location tumor:		not applicable

tag: TNM stage:			not applicable

tag: FEV1/FVC:			29 %


title: Ex-smoker, Squamous cell lung carcinoma T08 (smoke)

source_name: T08, ex-smoker

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Age at time of surgery:	54

tag: Packyears:			35

tag: Type of Surgery:		wedge resection

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		left upper lobe

tag: Tumor stage:			IA

tag: Dead/alive at last follow-up:	alive

tag: Survival as from surgery:	97 months

tag: Metastases:			no

tag: Time to distant metastasis:	not applicable

tag: COPD:				no COPD

tag: FEV1/FVC:			71 %

tag: GOLD stage:			not applicable


title: Ex-smoker, Normal B28 (smoke)

source_name: B28, ex-smoker

tag: Sample code

tag: Cell type:			histologically normal bronchial epithelial cells that were laser microdissected from frozen bronchial wall tissue

tag: Gender

tag: Age at time of surgery:	55

tag: Smoking status:		ex

tag: Packyears:			39

tag: Surgery due to:		lung transplantation

tag: Type of Surgery:		lung-explantation

tag: Location tumor:		not applicable

tag: COPD:				COPD

tag: FEV1/FVC:			21 %

tag: GOLD stage:			GOLD IV


title: Ex-smoker, Normal B26 (smoke)

source_name: B26, ex-smoker

tag: Age at time of surgery:	52

tag: Packyears:			18

tag: TNM stage:			not applicable

tag: FEV1/FVC:			33 %


title: Current-smoker, Squamous cell lung carcinoma T03 (smoke)

data_processing: Quantile normalization was applied to log2 transformed median signals of Cy5 and Cy3 intensities. Principal component analysis was performed and samples with a factor loading with a first principal component of more than two times the standard deviation from the mean were excluded as their hybridisations were considered to be of low quality. For this sample T03, dataset of Cy5 was excluded. Intensities were standardised by subtracting the variance explained by the first principal component, as this is gene specific variation that does not contribute to differences between arrays. Probe sequences that were spotted more than once were averaged.

source_name: T03, current smoker

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Age at time of surgery:	53

tag: Smoking status:		current

tag: Packyears:			40

tag: Type of Surgery:		lobectomy

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		left lower lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Dead/alive at last follow-up:	alive

tag: Survival as from surgery:	173 months

tag: Metastases:			no

tag: Time to distant metastasis:	not applicable

tag: COPD:				no COPD

tag: FEV1/FVC:			71 %

tag: GOLD stage:			not applicable


title: Current-smoker, Squamous cell lung carcinoma T01 (smoke)

source_name: T01, current smoker

tag: Sample code

tag: Gender

tag: Age at time of surgery:	67

tag: Packyears:			30

tag: TNM stage:			T2N1M0

tag: Tumor stage:			IIB

tag: Survival as from surgery:	80 months

tag: Metastases:			lymph node

tag: FEV1/FVC:			80 %


title: Ex-smoker, Normal B23 (smoke)

data_processing: Quantile normalization was applied to log2 transformed median signals of Cy5 and Cy3 intensities. Principal component analysis was performed and samples with a factor loading with a first principal component of more than two times the standard deviation from the mean were excluded as their hybridisations were considered to be of low quality. For this sample B23, dataset of Cy5 was excluded. Intensities were standardised by subtracting the variance explained by the first principal component, as this is gene specific variation that does not contribute to differences between arrays. Probe sequences that were spotted more than once were averaged.

source_name: B23, ex-smoker

tag: Cell type:			histologically normal bronchial epithelial cells that were laser microdissected from frozen bronchial wall tissue

tag: Age at time of surgery:	57

tag: Smoking status:		ex

tag: Surgery due to:		lung transplantation

tag: Type of Surgery:		lung-explantation

tag: Location tumor:		not applicable

tag: TNM stage:			not applicable

tag: COPD:				COPD

tag: FEV1/FVC:			23 %

tag: GOLD stage:			GOLD IV


title: Ex-smoker, Normal B24 (smoke)

source_name: B24, ex-smoker

tag: Sample code

tag: Gender

tag: Packyears:			25

tag: FEV1/FVC:			29 %


title: Ex-smoker, Normal B27 (smoke)

source_name: B27, ex-smoker

tag: Age at time of surgery:	58

tag: Packyears:			80

tag: FEV1/FVC:			49 %


title: Current-smoker, Squamous cell lung carcinoma T02 (smoke)

source_name: T02, current smoker

tag: Cell type:			tumor cells that were laser microdissected from frozen squamous cell lung carcinoma tissue

tag: Age at time of surgery:	67

tag: Smoking status:		current

tag: Type of Surgery:		lobectomy

tag: Tumor type:			squamous cell lung carcinoma

tag: Location tumor:		left upper lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Dead/alive at last follow-up:	alive

tag: Survival as from surgery:	151 months

tag: Metastases:			no

tag: Time to distant metastasis:	not applicable

tag: COPD:				no COPD

tag: FEV1/FVC:			71 %

tag: GOLD stage:			not applicable


title: Ex-smoker, Normal B21 (smoke)

source_name: B21, ex-smoker

tag: Sample code

tag: Cell type:			histologically normal bronchial epithelial cells that were laser microdissected from frozen bronchial wall tissue

tag: Gender

tag: Age at time of surgery:	54

tag: Smoking status:		ex

tag: Packyears:			31

tag: Surgery due to:		lung transplantation

tag: Type of Surgery:		lung-explantation

tag: Location tumor:		not applicable

tag: TNM stage:			not applicable

tag: COPD:				COPD

tag: FEV1/FVC:			38 %

tag: GOLD stage:			GOLD IV


title: WK1-E vs. WK1-placebo (REP1, HYB2)

status: Public on Dec 01 2008

city: RTP

first: NIEHS

last: Microarray Core

state: NC

address: 111 T.W. Alexander Drive

department: DIR

institute: NIEHS

description: Frozen mammary gland tissue, collected from four to five mice at day seven (placebo) or day seven (estradiol), was pooled and RNA was prepared using Trizol reagent and the RNeasy clean up protocol.

data_processing: Five hundred ng of total RNA was amplified and labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit according to the manufacturer's protocol.  For each two color comparison, 750 ng of each Cy3- and Cy5-labeled cRNA were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol.  Hybridizations were performed for 17 hours in a rotating hybridization oven according to the Agilent 60-mer oligo microarray processing protocol prior to washing and scanning with an Agilent Scanner.  Data was obtained using the Agilent Feature Extraction software (v7.5), using default for all parameters.

organism: Mus musculus

source_name: WK1-E (Cy3)

source_name: WK1-placebo (Cy5)


title: Current-smoker, Normal B19 (smoke)

source_name: B19, current smoker

tag: Age at time of surgery:	66

tag: Smoking status:		current

tag: Packyears:			50

tag: Surgery due to:		squamous cell carcinoma

tag: Type of Surgery:		lobectomy

tag: Location tumor:		left upper lobe

tag: TNM stage:			T3N0M0

tag: FEV1/FVC:			58 %

tag: GOLD stage:			GOLD II


title: Current-smoker, Normal B16 (smoke)

source_name: B16, current smoker

tag: Sample code

tag: Cell type:			histologically normal bronchial epithelial cells that were laser microdissected from frozen bronchial wall tissue

tag: Gender

tag: Age at time of surgery:	51

tag: Packyears:			60

tag: Type of Surgery:		pneumectomy

tag: TNM stage:			T3N1M0

tag: FEV1/FVC:			61 %

tag: GOLD stage:			GOLD I


title: Current-smoker, Normal B14 (smoke)

source_name: B14, current smoker

tag: Age at time of surgery:	53

tag: Smoking status:		current

tag: Packyears:			40

tag: Location tumor:		left lower lobe

tag: TNM stage:			T2N0M0

tag: COPD:				no COPD

tag: FEV1/FVC:			71 %

tag: GOLD stage:			not applicable


title: Current-smoker, Normal B17 (smoke)

source_name: B17, current smoker

tag: Sample code

tag: Age at time of surgery:	60

tag: Surgery due to:		squamous cell carcinoma

tag: Type of Surgery:		lobectomy

tag: TNM stage:			T2N1M0

tag: COPD:				COPD


title: Current-smoker, Normal B12 (smoke)

source_name: B12, current smoker

tag: Cell type:			histologically normal bronchial epithelial cells that were laser microdissected from frozen bronchial wall tissue

tag: Gender

tag: Age at time of surgery:	52

tag: Packyears:			20

tag: Type of Surgery:		bilobectomy

tag: Location tumor:		right lower lobe

tag: FEV1/FVC:			75 %


title: Current-smoker, Normal B09 (smoke)

source_name: B09, current smoker

tag: Age at time of surgery:	72

tag: Smoking status:		current

tag: Packyears:			52

tag: Type of Surgery:		pneuectomy

tag: Location tumor:		left upper lobe


title: Ex-smoker, Normal B11 (smoke)

source_name: B11, ex-smoker

tag: Sample code

tag: Age at time of surgery:	65

tag: Smoking status:		ex

tag: Packyears:			26

tag: Type of Surgery:		pneumectomy

tag: Location tumor:		right upper lobe

tag: TNM stage:			T2N0M0

tag: COPD:				no COPD

tag: FEV1/FVC:			79 %

tag: GOLD stage:			not applicable


title: Expression of S. agalactiae NEM316 genes in THY in early stationary phase (replicate 1)

city: Warsaw

first: Izabela

last: Sitkiewicz

address: Chełmska 30/34

department: Epidemiology and Clinical Microbiology

country: Poland

institute: National Medicines Institute

description: Analysis of S. agalactiae gene expression in laboratory medium

hyb_protocol: Overnight at 45C, standard Affymetrix protocol

scan_protocol: Affymetrix GeneChip scanner 3000

data_processing: Raw data normalized to total signal intensity of non redundant S. agalactiae probes present on the array

organism: Streptococcus agalactiae

source_name: S. agalactiae, 37 degrees at early stationary phase (replicate 1)

label_protocol: Standard Affymetrix

extract_protocol: 5ml culture was added to 10ml RNA protect (Qiagen), and GBS collected by centrifugation and stored at -80C. RNA was extracted from bacterial cell pellets through mechanical disruption using glass beads (MatrixB tubes) in a FastPrep machine. RNA was purified using TRIZOL extraction and re-purified on Qiagen miRNeasy columns using standard protocol. DNA was removed with DNA-free (Ambion). RNA quality was assessed by bioanalyzer and O.D. measurements.

tag: strain NEM316 grown in THY broth


title: Current-smoker, Normal B18 (smoke)

source_name: B18, current smoker

tag: Cell type:			histologically normal bronchial epithelial cells that were laser microdissected from frozen bronchial wall tissue

tag: Gender

tag: Age at time of surgery:	46

tag: Packyears:			25

tag: Surgery due to:		squamous cell carcinoma

tag: COPD:				COPD

tag: FEV1/FVC:			51 %

tag: GOLD stage:			GOLD II


title: Ex-smoker, Normal B13 (smoke)

source_name: B13, ex-smoker

tag: Sample code

tag: Age at time of surgery:	68

tag: Smoking status:		ex

tag: Packyears:			45

tag: Location tumor:		left upper lobe

tag: TNM stage:			T2N1M0

tag: FEV1/FVC:			77 %


title: Expression of S. agalactiae NEM316 genes in THY in late log phase (replicate 2)

source_name: S. agalactiae, 37 degrees at late log phase (replicate 2)


title: Expression of S. agalactiae NEM316 genes in THY in stationary phase (replicate 3)

source_name: S. agalactiae, 37 degrees at stationary phase (replicate 3)


title: Expression of S. agalactiae NEM316 genes in THY in stationary phase (replicate 2)

source_name: S. agalactiae, 37 degrees at stationary phase (replicate 2)


title: Expression of S. agalactiae NEM316 genes in THY in stationary phase (replicate 1)

source_name: S. agalactiae, 37 degrees at stationary phase (replicate 1)


title: Ex-smoker, Normal B15 (smoke)

source_name: B15, ex-smoker

tag: Age at time of surgery:	76

tag: Packyears:			40

tag: Type of Surgery:		lobectomy

tag: Location tumor:		left lower lobe

tag: TNM stage:			T1N0M0

tag: FEV1/FVC:			59 %

tag: GOLD stage:			GOLD I


title: Expression of S. agalactiae NEM316 genes in THY in early stationary phase (replicate 2)

source_name: S. agalactiae, 37 degrees at early stationary phase (replicate 2)

tag: strain NEM316 grown in THY broth


title: Expression of S. agalactiae NEM316 genes in THY in late log phase (replicate 1)

source_name: S. agalactiae, 37 degrees at late log phase (replicate 1)


title: Ex-smoker, Normal B20 (smoke)

source_name: B20, ex-smoker

tag: Cell type:			histologically normal bronchial epithelial cells that were laser microdissected from frozen bronchial wall tissue

tag: Gender

tag: Age at time of surgery:	73

tag: Surgery due to:		squamous cell carcinoma

tag: COPD:				COPD

tag: FEV1/FVC:			63 %


title: Expression of S. agalactiae NEM316 genes in THY in early stationary phase (replicate 3)

source_name: S. agalactiae, 37 degrees at early stationary phase (replicate 3)


title: Expression of S. agalactiae NEM316 genes in THY in mid log phase (replicate 3)

source_name: S. agalactiae, 37 degrees at mid log phase (replicate 3)


title: Expression of S. agalactiae NEM316 genes in THY in mid log phase (replicate 2)

source_name: S. agalactiae, 37 degrees at mid log phase (replicate 2)


title: Ex-smoker, Normal B05 (smoke)

source_name: B05, ex-smoker

tag: Sample code

tag: Smoking status:		ex

tag: Packyears:			27

tag: Surgery due to:		broncho alveolar carcinoma

tag: Location tumor:		left upper lobe

tag: TNM stage:			T2N0M0

tag: FEV1/FVC:			37 %

tag: GOLD stage:			GOLD II


title: Expression of S. agalactiae NEM316 genes in THY in late log phase (replicate 3)

source_name: S. agalactiae, 37 degrees at late log phase (replicate 3)


title: Current-smoker, Normal B08 (smoke)

source_name: B08, current smoker

tag: Age at time of surgery:	67

tag: Smoking status:		current

tag: Packyears:			13

tag: Type of Surgery:		lobectomy

tag: Location tumor:		right lower lobe

tag: TNM stage:			T1N0M0

tag: FEV1/FVC:			60 %


title: Current-smoker, Normal B03 (smoke)

source_name: B03, current smoker

tag: Cell type:			histologically normal bronchial epithelial cells that were laser microdissected from frozen bronchial wall tissue

tag: Gender

tag: Age at time of surgery:	57

tag: Packyears:			40

tag: Surgery due to:		adenocarcinoma

tag: TNM stage:			T1N2M0

tag: COPD:				COPD

tag: FEV1/FVC:			58 %

tag: GOLD stage:			GOLD I


title: Current-smoker, Normal B04 (smoke)

source_name: B04, current smoker

tag: Sample code

tag: Age at time of surgery:	63

tag: Location tumor:		left upper lobe

tag: FEV1/FVC:			39 %

tag: GOLD stage:			GOLD II


title: Ex-smoker, Normal B07 (smoke)

source_name: B07, ex-smoker

tag: Age at time of surgery:	67

tag: Smoking status:		ex

tag: Packyears:			20

tag: Surgery due to:		large cell carcinoma

tag: Type of Surgery:		lobectomy

tag: Location tumor:		left lower lobe

tag: COPD:				no COPD

tag: FEV1/FVC:			81 %

tag: GOLD stage:			not applicable


title: Ex-smoker, Normal B02 (smoke)

scan_protocol: The microarray slides were washed following the user manual instructions and scanned using the Agilent dual laser DNA microarray scanner.

source_name: B02, ex smoker

tag: Cell type:			histologically normal bronchial epithelial cells that were laser microdissected from frozen bronchial wall tissue

tag: Gender

tag: Age at time of surgery:	59

tag: Packyears:			30

tag: Surgery due to:		adenocarcinoma

tag: Location tumor:		right lower lobe

tag: TNM stage:			T1N0M0

tag: FEV1/FVC:			71 %


title: Ex-smoker, Normal B06 (smoke)

source_name: B06, ex-smoker

tag: Sample code

tag: Age at time of surgery:	58

tag: Packyears:			25

tag: Location tumor:		right upper lobe

tag: FEV1/FVC:			74 %


title: Current-smoker, Normal B01 (smoke)

source_name: B01, current smoker

tag: Age at time of surgery:	52

tag: Smoking status:		current

tag: Packyears:			35

tag: Type of Surgery:		lobectomy

tag: TNM stage:			T2N0M0

tag: FEV1/FVC:			78 %


title: Expression of S. agalactiae NEM316 genes in THY in mid log phase (replicate 1)

source_name: S. agalactiae, 37 degrees at mid log phase (replicate 1)

tag: strain NEM316 grown in THY broth


title: T31(aCGH)

description: T31(aCGH): squamous cell lung carcinoma, lymph node metastasis, COPD versus reference sample

hyb_protocol: AMOUNT OF HYBRIDISATION-MIX Take note of the area of the array and establish the size of the lifterslip and the amount of necessary (pre-)hybridisation mix: Lifterslip	Volume hybridisation-mix 22 x 22 mm	30 µl 22 x 30 mm	50 µl 22 x 60 mm	100 µl This protocol is intended for 50 µl hybridisation mix. If necessary take in to account that the amount of labelled product should be increased or decreased. PREHYBRIDISATION - Block the array slide according to the protocol of the manufacturer. - Prepare the prehybridisation-mix by adding together (per 50 µl prehybrydisation-mix): 50 µl salmon sperm DNA (~10g/l), 2 µl 3M NaAc pH5.3, 100 µl ethanol - Mix the solution and store it in the freezer for 15 min. - Collect the precipitate by centrifugation at 14000 rpm for 10 min. at 4 oC in the eppendorf centrifuge. - Discard the supernatant and wash the pellet with 500 µl 70% ethanol. - Centrifuge for 5 min. at 14000 rpm. - Discard the supernatant, dry the pellet and dissolve it in 50 µl hybridisation mix with 5% dextran sulphate - Apply the necessary amount of prehybridisation-mix to the array, on an heated block and place a fresh ethanol cleaned lifterslip on top of the array, without introducing air-bubbles. - Prehybridise for 2 hours in a waterbath at 65oC using a hybridisation chamber. - Wash off the lifterslip with milliQ-water, and spin dry the array-slide quickly at 800 rpm for 3 min in the Mistral MSE centrifuge. PREPARING THE HYBRIDISATION-MIX (For 50 µl of hybridisation mix) - Add together: 200 µl COT1-DNA (1µg/µl), 10 µl 3M NaAc pH5.3, 470 µl ethanol - Mix the solution and store it in the freezer for 15 min. - Collect the precipitate by centrifugation at 14000 rpm for 10 min. in the eppendorf centrifuge. - Discard the supernatant and wash the pellet with 500 µl 70% ethanol. - Centrifuge for 5 min. at 14000 rpm. - Discard the supernatant, dry the pellet and dissolve it in 50 µl hybridisation mix with 5% dextran sulphate. Perform next steps as much in the dark as possible! LABELING The random primed labelling procedure is carried out with components from the BioPrime DNA Labelling System (Invitrogen). - Mix 600 ng of genomic DNA with 20 µl of 2.5x random primer solution in a 200 µl vial and make up the volume to 42 µl with MilliQ-water - Denature the DNA by heating the mixture at 99oC for 10 min. in the MJ-Cycler. Briefly centrifuge and place on ice for 10 min. - Add: 5 µl10xdNTP mixture, 2 µl Cyanine 3-dUTP or Cyanine 5-dUTP, 1 µl Klenow DNA polymerase - Mix well and incubate the vials for 16 hours in the MJ-cycler at 37 oC. - Pre-spin a MicroSpin G50 Column at 750g in the Eppendorf centrifuge according to the users manual of the columns. - Pool the labelled test and reference DNA-samples and amply this mixture on top of the pre-spinned column. Centrifugate the column for 2 min. at 750g and collect the purified labeled DNA in a fresh tube. - Apply the collected eluate to a Microcon YM-30 filter and centrifugate at 14000 rpm in the eppendorf centrifuge, until the volume is decreased to approximately 5 µl. (app. 5 min.) - Apply 50 µl of the prepared hybridisation mix to the filter and incubate for 10 min. - Collect the labelled DNA in hybridisation-mixture by inverting the filter into a fresh 1.5ml tube and spin for 2 min. at 9000 rpm. The eluate should show a purple colour. HYBRIDISATION - Denature the DNA in the hybridisation mixture at 100 oC for 5 min. - Apply the suitable amount of the denatured mix to the array, on a heated block; place a fresh, ethanol cleaned, lifterslip on top of the array without introducing air bubbles. - Hybridise for ~40 hours in the Gemini Twin shaker waterbath at 65oC using a hybridisation chamber. POST HYBRIDISATION STEPS - Transfer the slide to a washing rack - Wash the slides using the washing solutions described in the Schott Nexterion guide: 10 min. washing solution 1 at 65oC, 5 min. washing solution 2 at 20oC, 5 min. washing solution 3 at 20oC - Spin dry the array-slide quickly at 800 rpm for 3 min in the Mistral MSE centrifuge. - Scan the array using the Agilent scanner with optimal settings according to the manual. INTERNAL CONTROL Use a reference-DNA sample with opposite gender for these hybridisations. Check the altered ratio’s after a calculation procedure. On the array’s control spots are present, derived from Total Human DNA, CoT-DNA and Drosophila DNA. The intensities of these spots were taken into account in the calculation procedure.

scan_protocol: Agilent scanner (resolution 5 um) in combination with Agilent software. Default settings and protocols as supplied by Agilent

data_processing:
The scanned images were processed with Bluefuse v3.5 (BlueGnomeLtd, Cambridge, UK). A block-median normalization excluding controls was applied to the 2log-transformed Cy3/Cy5 ratios. Subsequent exclusion criteria were: confidence <0.3, quality <1 and SD of replicates >0.5. Log-ratios of replicates were combined using the fusion algorithm. The resulting log-ratios were imported into Nexus-software Copy Number-V3.1 (BioDiscovery, US) to visualise and analyse copy number changes within or between subsets of SCC.
The confidence flag filter selection: A and B (= confidence > 0.7)
Postprocessing protocol:
- Normalization then exclusion
- Block median normalization excluding controls
Next, exclude spots if:
- ID = controls, THEN
- Confidence < 0.3, THEN
- Quality < 1, THEN
- Replicates with StdDEV > 0.5
Next:
- Replicates are combined based on “Name”, using “Fusion” algorithm
Remark: Although this software package includes algorithms that deal with the (local) background, the background values are not listed in any of the output files.

source_name: lymph node metastasis, COPD

extract_protocol:
LASER DISSECTION MICROSCOPY
- Frozen tumor sections of 8μm were used for laser dissection microscopy (LDM)
- Only vital tumor cells without apparent admixture of inflammatory cells through the tumor fields were isolated using laser dissection microscopy
- An area of approximately 25x106 μm2 was microdissected from the section using P.A.L.M. Microlaser Technology system (P.A.L.M., Bernried, Germany).
- Laser microdissected cells were collected in 20 μl SE buffer
DNA ISOLATION USING A STANDARD SALT-CHLOROFORM EXTRACTION PROTOCOL:
- Add 400 μl SE buffer, 20 μl 10% SDS and 2 μl Proteinase K (20 mg/ml)
- Incubate the solution overnight at 55°C in the incubator; also incubate the saturated NaCl solution (6M) overnight at 55°C
- Add 134 μl preheated saturated NaCl solution to the solution
- Add 1 volume (556 μl) chloroform
- Mix 1 hour on a top-over-top rotor
- Centrifuge for 2 minutes, 15000 rpm, 4°C
- Transfer upper phase to a clean tube
- Add 1 volume isopropanol and mix carefully
- Centrifuge for 2 minutes, 15000 rpm, 4°C
- Wash pellet/cloud with 200 μl 80% ethanol
- Centrifuge for 2 minutes, 15000 rpm, 4°C
- Pipette off all the ethanol, shortly centrifuge and pipette off again
- Dry pellet on air for 5-10 minutes
- Dissolve pellet in 20 μl TE-4
- The DNA concentration was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE)
For each sample, 100 ng genomic DNA was amplified using the BioScore kit (Enzo Life Sciences, Farmingdale, NY):
AMPLIFYING THE GENOMIC DNA
In order to establish the lower and upper limits of the amplification reaction, a negative control reaction (no template DNA) and a positive control reaction (100 ng of Excellent high quality, non-FFPE genomic DNA) should be included in the protocol. We recommend amplifying the same reference DNA that will be labeled for array analysis (e.g., pooled normal male or pooled normal female from Promega).
1. Combine 100 ng (up to 19 μl) of genomic DNA (FFPE-extracted or non-FFPE) with 20 μl Primers (P) and add enough Nuclease-free Water (W) to bring the reaction mixture to 39 μl. Mix by gently flicking tube, centrifuge briefly.
2. Heat at 99oC for 10 minutes. Place tubes immediately on ice for 5 minutes. Centrifuge briefly and return to ice.
3. While on ice, add 10 μl Nucleotide Mix (N) and 1 μl Enzyme (E). Mix by gently flicking the tube. Centrifuge briefly and incubate at 37oC for 1 hour.
4. Add 5 μl of Stop Buffer (S). Mix by gently flicking the tube. Centrifuge briefly and keep at room temperature.
PURIFYING THE AMPLIFIED DNA
1. Use the QIAquick PCR Purification Kit. Add 300 μl of PB Buffer (≥ 5 volumes) to each tube. Mix thoroughly by repeated inversion and centrifuge briefly. Add each sample to a separate QIAquick column. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate.
2. Add 750 μl of Buffer PE. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate
3. Add 750 μl of Buffer PE. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate. Transfer the QIAquick column to a new collection tube. Centrifuge again at 13,000 RPM (16,000 x g) for 1 minute. Transfer the QIAquick column to a new 1.5 ml microfuge tube.
4. Add 25 μl of Buffer EB (preheated to 37oC). Incubate at RT for 1 minute. Elute the amplified DNA by centrifugation at 13,000 RPM (16,000 x g) for 1 minute. Add an additional 25 μl of Buffer EB (preheated to 37oC). Incubate at RT for 1 minute. Centrifuge at 13,000 RPM (16,000 x g).
5. Measure concentration of the amplified DNA by spectrophotometric analysis and calculate yield.
6. Store amplified products at 4°C for short-term storage (< 1 week) and at –20oC for long-term storage.
INTERPRETING THE RESULTS OF THE AMPLIFICATION REACTION
The table below indicates the DNA yields expected from a 1 hour amplification of 100 ng of input DNA. These values are not applicable for reactions longer than 1 hour or with inputs greater than or less than 100 ng. Samples with yields in the Poor range are unlikely to yield informative array results and should not be used.
DNA Quality	Total Yield (ug)
No input	<0.5
Poor		<1
Intermediate	1-3
Good		3-10
Excellent	>10
The exact yields used for scoring DNAs may vary slightly depending on the user’s labeling conditions and array platform.
LABELING FOR ARRAY ASSAYS
1. Direct Labeling of Unamplified DNA
- 0.85 μg of Good genomic DNA can be used for direct labeling and is likely to yield excellent array results.
- >0.85 μg of Intermediate genomic DNA should be used for direct labeling (e.g., 1.7 μg).
- Poor input DNA should not labeled.
2. Labeling of Amplified DNA
- 0.85 μg of amplified DNA from 100 ng of genomic sample DNAs scored as Excellent or Good can be labeled for successful array analysis.
- 0.85 μg of amplified DNA from from 100 ng of genomic sample DNAs scored as Intermediate can be labeled for array analysis. These arrays, however, are likely to have higher noise levels than arrays using unamplified DNA.
Only samples scored for Excellent or Good were used for further processing.

tag: Tissue type

value: Human lung tumor (frozen)

tag: Gender

value: Female

tag: Age at time of surgery:	61

tag: Tumor type

value: Squamous cell lung carcinoma (SCC)

tag: Type of Surgery:		Pneumectomy

tag: Location tumor:		Right upper lobe

tag: TNM stage:			T2N1M0

tag: Tumor stage:			IIB

tag: Metastases:			lymph node

tag: Time to distant metastases as from surgery (months)	:	not applicable

tag: Location of distant metastasis:				not applicable

tag: Dead/Alive:							alive

tag: Survival as from SCC resection (months):			85

tag: COPD:				COPD

tag: FEV1/FVC:			65 %

tag: GOLD stage:			GOLD I

source_name: Reference sample

label_protocol:
Prior to the hybridisation, the genomic "test" and "reference" DNA are labelled with Cy3 and Cy5 fluorescent dyes respectively. The random primed labelling procedure is carried out with components from the BioPrime DNA Labelling System (Invitrogen).
- Mix 600 ng of genomic DNA with 20 µl of 2.5x random primer solution in a 200 µl vial and make up the volume to 42 µl with MilliQ-water
- Denature the DNA by heating the mixture at 99oC for 10 min. in the MJ-Cycler.
Briefly centrifuge and place on ice for 10 min.
- Add: 5 µl10xdNTP mixture, 2 µl Cyanine 3-dUTP or Cyanine 5-dUTP, 1 µl Klenow DNA polymerase
- Mix well and incubate the vials for 16 hours in the MJ-cycler at 37 oC.
- Pre-spin a MicroSpin G50 Column at 750g in the Eppendorf centrifuge according to the users manual of the columns.
- Pool the labelled test and reference DNA-samples and amply this mixture on top of the pre-spinned column. Centrifugate the column for 2 min. at 750g and collect the purified labeled DNA in a fresh tube.
- Apply the collected eluate to a Microcon YM-30 filter and centrifugate at 14000 rpm in the eppendorf centrifuge, until the volume is decreased to approximately 5 µl. (app. 5 min.)
- Apply 50 µl of the prepared hybridisation mix to the filter and incubate for 10 min.
- Collect the labelled DNA in hybridisation-mixture by inverting the filter into a fresh 1.5ml tube and spin for 2 min. at 9000 rpm. The eluate should show a purple colour.

extract_protocol:
Whole blood was collected in disodium EDTA containers and stored at -20ºC and a number of samples were also stored at -80ºC. To facilitate haemolysis of RBCs it is recommended to store a fresh sample for a few hours in a freezer as freezing destroys the red cells. After thawing, 3mls of whole blood are transferred to a sterile conical centrifuge tube (15ml volume) to which 9mls of 1 x erythrocyte lysing buffer (0.155M NH4Cl; 10mM KHCO3; 0.1mM Na2 EDTA; pH 7.4) must be added. The solution is left for 10 minutes at RT with occasional mixing by inversion followed by centrifugation for 5 minutes at 4000 rpm. After centrifugation the supernatant is discarded and a white pellet will be observed at the bottom of the tube. This pellet must be washed for at least 3 times by adding 3mls of erythrocyte lysing buffer and repeating steps 3 and 4. It is important to breakdown the pellet and rinse it well in erythrocyte lysing buffer in order to clean the white blood cells from remaining heme. 1.5mls of SE buffer (75mM NaCl; 25mM Na2 EDTA; pH 8.0) containing 100mg/ml of Proteinase K and 1% sodium dodecyl sulphate (SDS w/v), are added to the pellet. The tubes are then incubated at a temperature of 37-55ºC (optimal temp for Proteinase K activity) overnight in a water bath or incubator. During this step the white blood cells' membranes are denatured and DNA goes out in solution. After the incubation, 1.5mls of SE buffer together with 750ml of 6M (saturated) NaCl are added to each tube, followed by the addition of 3.75mls chloroform. The tubes are mixed vigorously (on a vortex) for about 20 sec with occasional mixing for at least 30min. Alternatively you can leave the tubes on a rotator for 1 hour. The emulsion will then be centrifuged for 10 minutes at 2000 rpm with minimal breaking force. After centrifugation 2 phases are observed and care must be taken not to disturb the interphase. During this step DNA is extracted into the supernatant and proteins separated into the lower phase. The upper aqueous phase (containing the DNA) is transferred into a clean and sterile conical centrifuge tube using a sterile Pasteur pipette, followed by the addition of an equal volume of isopropanol. DNA will be precipitated by gentle swirling of the tube and is observed visually as a white thread like strand. Using a sterile spatula or loop transfer the DNA strand into a sterile microcentrifuge tube containing 1ml of 75% ethanol. The DNA is then washed by inversion to clean it from any remaining salts and the tube centrifuged at 11000g for 4 minutes. The supernatant is discarded taking care not to discard the pellet. Repeat this step once more. After discarding the supernatant the pellet is dried from excess ethanol either by using a vacuum centrifuge or by leaving the tubes open and inverted in an oven at around 50 - 65ºC for an hour. The dried pellet is resuspended in TE buffer (1M Tris-HCl; 0.5M EDTA; pH 8.0) and left overnight on a rotator. DNA concentration is determined either by agarose gel electrophoresis or spectrophotometry and adjusted to the desired concentration by adding more TE buffer. It is important to note that before adjusting and reading DNA concentrations one must obtain a homogeneous sample of DNA which is not quite easy acquired since DNA is very viscous. To adjust to lower concentration one must used other quantitation methods such as Picogreen (Molecular Probes) using spectrofluorometry as spectrophotometry is not accurate and sensitive at very low concentrations.
For each sample, 100 ng genomic DNA was amplified using the BioScore kit (Enzo Life Sciences, Farmingdale, NY):
AMPLIFYING THE GENOMIC DNA
In order to establish the lower and upper limits of the amplification reaction, a negative control reaction (no template DNA) and a positive control reaction (100 ng of Excellent high quality, non-FFPE genomic DNA) should be included in the protocol. We recommend amplifying the same reference DNA that will be labeled for array analysis (e.g., pooled normal male or pooled normal female from Promega).
1. Combine 100 ng (up to 19 μl) of genomic DNA (FFPE-extracted or non-FFPE) with 20 μl Primers (P) and add enough Nuclease-free Water (W) to bring the reaction mixture to 39 μl. Mix by gently flicking tube, centrifuge briefly.
2. Heat at 99oC for 10 minutes. Place tubes immediately on ice for 5 minutes. Centrifuge briefly and return to ice.
3. While on ice, add 10 μl Nucleotide Mix (N) and 1 μl Enzyme (E). Mix by gently flicking the tube. Centrifuge briefly and incubate at 37oC for 1 hour.
4. Add 5 μl of Stop Buffer (S). Mix by gently flicking the tube. Centrifuge briefly and keep at room temperature.
PURIFYING THE AMPLIFIED DNA
1. Use the QIAquick PCR Purification Kit. Add 300 μl of PB Buffer (≥ 5 volumes) to each tube. Mix thoroughly by repeated inversion and centrifuge briefly. Add each sample to a separate QIAquick column. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate.
2. Add 750 μl of Buffer PE. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate
3. Add 750 μl of Buffer PE. Centrifuge at 13,000 RPM (16,000 x g) for 1 minute. Discard filtrate. Transfer the QIAquick column to a new collection tube. Centrifuge again at 13,000 RPM (16,000 x g) for 1 minute. Transfer the QIAquick column to a new 1.5 ml microfuge tube.
4. Add 25 μl of Buffer EB (preheated to 37oC). Incubate at RT for 1 minute. Elute the amplified DNA by centrifugation at 13,000 RPM (16,000 x g) for 1 minute. Add an additional 25 μl of Buffer EB (preheated to 37oC). Incubate at RT for 1 minute. Centrifuge at 13,000 RPM (16,000 x g).
5. Measure concentration of the amplified DNA by spectrophotometric analysis and calculate yield.
6. Store amplified products at 4°C for short-term storage (< 1 week) and at –20oC for long-term storage.
INTERPRETING THE RESULTS OF THE AMPLIFICATION REACTION
The table below indicates the DNA yields expected from a 1 hour amplification of 100 ng of input DNA. These values are not applicable for reactions longer than 1 hour or with inputs greater than or less than 100 ng. Samples with yields in the Poor range are unlikely to yield informative array results and should not be used.
DNA Quality	Total Yield (ug)
No input	<0.5
Poor		<1
Intermediate	1-3
Good		3-10
Excellent	>10
The exact yields used for scoring DNAs may vary slightly depending on the user’s labeling conditions and array platform.
LABELING FOR ARRAY ASSAYS
1. Direct Labeling of Unamplified DNA
- 0.85 μg of Good genomic DNA can be used for direct labeling and is likely to yield excellent array results.
- >0.85 μg of Intermediate genomic DNA should be used for direct labeling (e.g., 1.7 μg).
- Poor input DNA should not labeled.
2. Labeling of Amplified DNA
- 0.85 μg of amplified DNA from 100 ng of genomic sample DNAs scored as Excellent or Good can be labeled for successful array analysis.
- 0.85 μg of amplified DNA from from 100 ng of genomic sample DNAs scored as Intermediate can be labeled for array analysis. These arrays, however, are likely to have higher noise levels than arrays using unamplified DNA.
Only samples scored for Excellent or Good were used for further processing.

tag: Mix of DNA from 20 healthy men


title: T26(aCGH)

description: T26(aCGH): squamous cell lung carcinoma, distant metastasis, COPD versus reference sample

source_name: distant metastasis, COPD

label_protocol:
The random primed labelling procedure is carried out with components from the BioPrime DNA Labelling System (Invitrogen).
- Mix 600 ng of genomic DNA with 20 µl of 2.5x random primer solution in a 200 µl vial and make up the volume to 42 µl with MilliQ-water
- Denature the DNA by heating the mixture at 99oC for 10 min. in the MJ-Cycler.
Briefly centrifuge and place on ice for 10 min.
- Add: 5 µl10xdNTP mixture, 2 µl Cyanine 3-dUTP or Cyanine 5-dUTP, 1 µl Klenow DNA polymerase
- Mix well and incubate the vials for 16 hours in the MJ-cycler at 37 oC.
- Pre-spin a MicroSpin G50 Column at 750g in the Eppendorf centrifuge according to the users manual of the columns.
- Pool the labelled test and reference DNA-samples and amply this mixture on top of the pre-spinned column. Centrifugate the column for 2 min. at 750g and collect the purified labeled DNA in a fresh tube.
- Apply the collected eluate to a Microcon YM-30 filter and centrifugate at 14000 rpm in the eppendorf centrifuge, until the volume is decreased to approximately 5 µl. (app. 5 min.)
- Apply 50 µl of the prepared hybridisation mix to the filter and incubate for 10 min.
- Collect the labelled DNA in hybridisation-mixture by inverting the filter into a fresh 1.5ml tube and spin for 2 min. at 9000 rpm. The eluate should show a purple colour.

tag: Sample code

tag: Tissue type

value: Male

tag: Age at time of surgery:	48

tag: Packyears:			25

tag: Type of Surgery:		Bilobectomy

tag: Location tumor:		Left lower lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Metastases:			distant

tag: Time to distant metastases as from surgery (months)	:	19

tag: Location of distant metastasis:				brain, kidney

tag: Dead/Alive:							dead

tag: Survival as from SCC resection (months):			34

tag: FEV1/FVC:			54 %

tag: GOLD stage:			GOLD II

tag: Mix of DNA from 20 healthy women


title: T33(aCGH)

description: T33(aCGH): squamous cell lung carcinoma, lymph node metastasis, COPD versus reference sample

tag: Gender

tag: Age at time of surgery:	51

tag: Packyears:			33

tag: Tumor type

tag: Type of Surgery:		Pneumectomy

tag: Location tumor:		Right middle lobe

tag: TNM stage:			T2N1M0

tag: Tumor stage:			IIB

tag: Metastases:			lymph node

tag: Time to distant metastases as from surgery (months)	:	not applicable

tag: Location of distant metastasis:				not applicable

tag: Dead/Alive:							alive

tag: Survival as from SCC resection (months):			69

tag: COPD:				COPD

tag: FEV1/FVC:			64 %

tag: GOLD stage:			GOLD I


title: T25(aCGH)

description: T25(aCGH): squamous cell lung carcinoma, distant metastasis, COPD versus reference sample

tag: Sample code

tag: Tissue type

tag: Age at time of surgery:	66

tag: Packyears:			50

tag: Type of Surgery:		Lobectomy

tag: Location tumor:		Left upper lobe

tag: TNM stage:			T3N0M0

tag: Metastases:			distant

tag: Time to distant metastases as from surgery (months)	:	11

tag: Location of distant metastasis:				kidney/adrenals

tag: Survival as from SCC resection (months):			20

tag: FEV1/FVC:			58 %

tag: GOLD stage:			GOLD II

tag: Mix of DNA from 20 healthy women


title: T29(aCGH)

description: T29(aCGH): squamous cell lung carcinoma, lymph node metastasis, no COPD versus reference sample

source_name: lymph node metastasis, no COPD

tag: Gender

tag: Age at time of surgery:	58

tag: Packyears:			10

tag: Tumor type

tag: Type of Surgery:		Pneumectomy

tag: Location tumor:		Right lower lobe

tag: TNM stage:			T2N2M0

tag: Tumor stage:			IIIA

tag: Metastases:			lymph node

tag: Time to distant metastases as from surgery (months)	:	not applicable

tag: Location of distant metastasis:				not applicable

tag: Dead/Alive:							alive

tag: Survival as from SCC resection (months):			130

tag: COPD:				no COPD

tag: FEV1/FVC:			71 %

tag: GOLD stage:			not applicable

tag: Mix of DNA from 20 healthy men


title: T27(aCGH)

description: T27(aCGH): squamous cell lung carcinoma, distant metastasis, COPD versus reference sample

tag: Sample code

tag: Tissue type

tag: Gender

tag: Age at time of surgery:	75

tag: Packyears:			40

tag: Type of Surgery:		Lobectomy

tag: Location tumor:		Left upper lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Metastases:			distant

tag: Time to distant metastases as from surgery (months)	:	2

tag: Location of distant metastasis:				brain

tag: Dead/Alive:							dead

tag: Survival as from SCC resection (months):			6

tag: COPD:				COPD

tag: FEV1/FVC:			66 %

tag: GOLD stage:			GOLD II

tag: Mix of DNA from 20 healthy women


title: T32(aCGH)

description: T32(aCGH): squamous cell lung carcinoma, lymph node metastasis, COPD versus reference sample

tag: Sample code

tag: Tissue type

tag: Age at time of surgery:	63

tag: Tumor type

tag: Type of Surgery:		Pneumectomy

tag: Location tumor:		Left lower lobe

tag: TNM stage:			T2N1M0

tag: Tumor stage:			IIB

tag: Metastases:			lymph node

tag: Time to distant metastases as from surgery (months)	:	not applicable

tag: Location of distant metastasis:				not applicable

tag: Survival as from SCC resection (months):			91

tag: FEV1/FVC:			62 %

tag: GOLD stage:			GOLD I


title: T22(aCGH)

description: T22(aCGH): squamous cell lung carcinoma, distant metastasis, no COPD versus reference sample

source_name: distant metastasis, no COPD

tag: Gender

tag: Age at time of surgery:	55

tag: Packyears:			30

tag: Location tumor:		Left upper lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Metastases:			distant

tag: Time to distant metastases as from surgery (months)	:	17

tag: Location of distant metastasis:				right lung

tag: Dead/Alive:							alive

tag: Survival as from SCC resection (months):			20

tag: COPD:				no COPD

tag: FEV1/FVC:			72 %

tag: GOLD stage:			not applicable


title: T30(aCGH)

description: T30(aCGH): squamous cell lung carcinoma, lymph node metastasis, no COPD versus reference sample

tag: Sample code

tag: Tissue type

tag: Age at time of surgery:	71

tag: Packyears:			3

tag: Tumor type

tag: Type of Surgery:		Pneumectomy

tag: Location tumor:		Right lower lobe

tag: TNM stage:			T2N1M0

tag: Tumor stage:			IIB

tag: Metastases:			lymph node

tag: Time to distant metastases as from surgery (months)	:	not applicable

tag: Location of distant metastasis:				not applicable

tag: Dead/Alive:							dead

tag: Survival as from SCC resection (months):			71

tag: Mix of DNA from 20 healthy men


title: T18(aCGH)

description: T18(aCGH): squamous cell lung carcinoma, distant metastasis, COPD versus reference sample

tag: Gender

tag: Age at time of surgery:	70

tag: Packyears:			36

tag: Type of Surgery:		Bilobectomy

tag: Location tumor:		Right upper lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Metastases:			distant

tag: Time to distant metastases as from surgery (months)	:	9

tag: Location of distant metastasis:				left lung

tag: Survival as from SCC resection (months):			23

tag: COPD:				COPD

tag: FEV1/FVC:			54 %

tag: GOLD stage:			GOLD I

tag: Mix of DNA from 20 healthy women


title: T23(aCGH)

description: T23(aCGH): squamous cell lung carcinoma, distant metastasis, no COPD versus reference sample

tag: Sample code

tag: Tissue type

tag: Age at time of surgery:	60

tag: Packyears:			40

tag: Tumor type

tag: Type of Surgery:		Lobectomy

tag: TNM stage:			T3N0M0

tag: Tumor stage:			IIB

tag: Time to distant metastases as from surgery (months)	:	6

tag: Dead/Alive:							dead

tag: Survival as from SCC resection (months):			9

tag: COPD:				no COPD

tag: FEV1/FVC:			73 %

tag: GOLD stage:			not applicable


title: T20(aCGH)

description: T20(aCGH): squamous cell lung carcinoma, distant metastasis, no COPD versus reference sample

tag: Gender

tag: Age at time of surgery:	71

tag: Packyears:			57

tag: Location tumor:		Left upper lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Metastases:			distant

tag: Time to distant metastases as from surgery (months)	:	28

tag: Location of distant metastasis:				bone, liver, right lung

tag: Survival as from SCC resection (months):			28

tag: FEV1/FVC:			75 %


title: T34(aCGH)

description: T34(aCGH): squamous cell lung carcinoma, lymph node metastasis, COPD versus reference sample

tag: Sample code

tag: Tissue type

tag: Age at time of surgery:	54

tag: Packyears:			30

tag: Tumor type

tag: Type of Surgery:		Pneumectomy

tag: Location tumor:		Right lower lobe

tag: TNM stage:			T3N1M0

tag: Tumor stage:			IIIA

tag: Metastases:			lymph node

tag: Time to distant metastases as from surgery (months)	:	not applicable

tag: Location of distant metastasis:				not applicable

tag: Dead/Alive:							alive

tag: Survival as from SCC resection (months):			128

tag: COPD:				COPD

tag: FEV1/FVC:			61 %

tag: GOLD stage:			GOLD II

tag: Mix of DNA from 20 healthy women


title: T19(aCGH)

description: T19(aCGH): squamous cell lung carcinoma, distant metastasis, no COPD versus reference sample

tag: Sample code

tag: Tissue type

tag: Gender

tag: Age at time of surgery:	71

tag: Packyears:			51

tag: Type of Surgery:		Lobectomy

tag: Location tumor:		Left upper lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Metastases:			distant

tag: Time to distant metastases as from surgery (months)	:	13

tag: Location of distant metastasis:				pericard, pleura

tag: Dead/Alive:							dead

tag: Survival as from SCC resection (months):			13

tag: COPD:				no COPD

tag: FEV1/FVC:			79 %

tag: GOLD stage:			not applicable


title: T24(aCGH)

description: T24(aCGH): squamous cell lung carcinoma, distant metastasis, COPD versus reference sample

tag: Sample code

tag: Age at time of surgery:	67

tag: Packyears:			45

tag: Tumor type

tag: Type of Surgery:		Pneumectomy

tag: Location tumor:		Right lower lobe

tag: Time to distant metastases as from surgery (months)	:	16

tag: Location of distant metastasis:				liver

tag: Survival as from SCC resection (months):			16

tag: COPD:				COPD

tag: FEV1/FVC:			63 %

tag: GOLD stage:			GOLD I

tag: Mix of DNA from 20 healthy women


title: T28(aCGH)

description: T28(aCGH): squamous cell lung carcinoma, lymph node metastasis, no COPD versus reference sample

extract_protocol:
DNA ISOLATION USING A STANDARD SALT-CHLOROFORM EXTRACTION PROTOCOL:
- Add 400 μl SE buffer, 20 μl 10% SDS and 2 μl Proteinase K (20 mg/ml)
- Incubate the solution overnight at 55°C in the incubator; also incubate the saturated NaCl solution (6M) overnight at 55°C
- Add 134 μl preheated saturated NaCl solution to the solution
- Add 1 volume (556 μl) chloroform
- Mix 1 hour on a top-over-top rotor
- Centrifuge for 2 minutes, 15000 rpm, 4°C
- Transfer upper phase to a clean tube
- Add 1 volume isopropanol and mix carefully
- Centrifuge for 2 minutes, 15000 rpm, 4°C
- Wash pellet/cloud with 200 μl 80% ethanol
- Centrifuge for 2 minutes, 15000 rpm, 4°C
- Pipette off all the ethanol, shortly centrifuge and pipette off again
- Dry pellet on air for 5-10 minutes
- Dissolve pellet in 20 μl TE-4
- The DNA concentration was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE)

tag: Tissue type

tag: Gender

tag: Age at time of surgery:	76

tag: Packyears:			40

tag: Location tumor:		Left upper lobe

tag: TNM stage:			T3N1M0

tag: Tumor stage:			IIIA

tag: Metastases:			lymph node

tag: Time to distant metastases as from surgery (months)	:	not applicable

tag: Location of distant metastasis:				not applicable

tag: Dead/Alive:							dead

tag: Survival as from SCC resection (months):			58

tag: FEV1/FVC:			73 %

label_protocol:
Prior to the hybridisation, the genomic "test" and "reference" DNA are labelled with Cy3 and Cy5 fluorescent dyes respectively. The random primed labelling procedure is carried out with components from the BioPrime DNA Labelling System (Invitrogen).
- Mix 250 ng of genomic DNA with 20 µl of 2.5x random primer solution in a 200 µl vial and make up the volume to 42 µl with MilliQ-water
- Denature the DNA by heating the mixture at 99oC for 10 min. in the MJ-Cycler.
Briefly centrifuge and place on ice for 10 min.
- Add: 5 µl10xdNTP mixture, 2 µl Cyanine 3-dUTP or Cyanine 5-dUTP, 1 µl Klenow DNA polymerase
- Mix well and incubate the vials for 16 hours in the MJ-cycler at 37 oC.
- Pre-spin a MicroSpin G50 Column at 750g in the Eppendorf centrifuge according to the users manual of the columns.
- Pool the labelled test and reference DNA-samples and amply this mixture on top of the pre-spinned column. Centrifugate the column for 2 min. at 750g and collect the purified labeled DNA in a fresh tube.
- Apply the collected eluate to a Microcon YM-30 filter and centrifugate at 14000 rpm in the eppendorf centrifuge, until the volume is decreased to approximately 5 µl. (app. 5 min.)
- Apply 50 µl of the prepared hybridisation mix to the filter and incubate for 10 min.
- Collect the labelled DNA in hybridisation-mixture by inverting the filter into a fresh 1.5ml tube and spin for 2 min. at 9000 rpm. The eluate should show a purple colour.

extract_protocol: Whole blood was collected in disodium EDTA containers and stored at -20ºC and a number of samples were also stored at -80ºC. To facilitate haemolysis of RBCs it is recommended to store a fresh sample for a few hours in a freezer as freezing destroys the red cells. After thawing, 3mls of whole blood are transferred to a sterile conical centrifuge tube (15ml volume) to which 9mls of 1 x erythrocyte lysing buffer (0.155M NH4Cl; 10mM KHCO3; 0.1mM Na2 EDTA; pH 7.4) must be added. The solution is left for 10 minutes at RT with occasional mixing by inversion followed by centrifugation for 5 minutes at 4000 rpm. After centrifugation the supernatant is discarded and a white pellet will be observed at the bottom of the tube. This pellet must be washed for at least 3 times by adding 3mls of erythrocyte lysing buffer and repeating steps 3 and 4. It is important to breakdown the pellet and rinse it well in erythrocyte lysing buffer in order to clean the white blood cells from remaining heme. 1.5mls of SE buffer (75mM NaCl; 25mM Na2 EDTA; pH 8.0) containing 100mg/ml of Proteinase K and 1% sodium dodecyl sulphate (SDS w/v), are added to the pellet. The tubes are then incubated at a temperature of 37-55ºC (optimal temp for Proteinase K activity) overnight in a water bath or incubator. During this step the white blood cells' membranes are denatured and DNA goes out in solution. After the incubation, 1.5mls of SE buffer together with 750ml of 6M (saturated) NaCl are added to each tube, followed by the addition of 3.75mls chloroform. The tubes are mixed vigorously (on a vortex) for about 20 sec with occasional mixing for at least 30min. Alternatively you can leave the tubes on a rotator for 1 hour. The emulsion will then be centrifuged for 10 minutes at 2000 rpm with minimal breaking force. After centrifugation 2 phases are observed and care must be taken not to disturb the interphase. During this step DNA is extracted into the supernatant and proteins separated into the lower phase. The upper aqueous phase (containing the DNA) is transferred into a clean and sterile conical centrifuge tube using a sterile Pasteur pipette, followed by the addition of an equal volume of isopropanol. DNA will be precipitated by gentle swirling of the tube and is observed visually as a white thread like strand. Using a sterile spatula or loop transfer the DNA strand into a sterile microcentrifuge tube containing 1ml of 75% ethanol. The DNA is then washed by inversion to clean it from any remaining salts and the tube centrifuged at 11000g for 4 minutes. The supernatant is discarded taking care not to discard the pellet. Repeat this step once more. After discarding the supernatant the pellet is dried from excess ethanol either by using a vacuum centrifuge or by leaving the tubes open and inverted in an oven at around 50 - 65ºC for an hour. The dried pellet is resuspended in TE buffer (1M Tris-HCl; 0.5M EDTA; pH 8.0) and left overnight on a rotator. DNA concentration is determined either by agarose gel electrophoresis or spectrophotometry and adjusted to the desired concentration by adding more TE buffer. It is important to note that before adjusting and reading DNA concentrations one must obtain a homogeneous sample of DNA which is not quite easy acquired since DNA is very viscous. To adjust to lower concentration one must used other quantitation methods such as Picogreen (Molecular Probes) using spectrofluorometry as spectrophotometry is not accurate and sensitive at very low concentrations.


title: T17(aCGH)

description: T17(aCGH): squamous cell lung carcinoma, distant metastasis, no COPD versus reference sample

tag: Sample code

tag: Age at time of surgery:	72

tag: Packyears:			60

tag: Tumor type

tag: Type of Surgery:		Bilobectomy

tag: Location tumor:		Right upper lobe

tag: TNM stage:			T3N0M0

tag: Tumor stage:			IIB

tag: Metastases:			distant

tag: Time to distant metastases as from surgery (months)	:	6

tag: Location of distant metastasis:				bone

tag: Dead/Alive:							alive

tag: Survival as from SCC resection (months):			15

tag: COPD:				no COPD

tag: FEV1/FVC:			77 %

tag: GOLD stage:			not applicable


title: T21(aCGH)

description: T21(aCGH): squamous cell lung carcinoma, distant metastasis, no COPD versus reference sample

tag: Tissue type

tag: Gender

tag: Age at time of surgery:	69

tag: Packyears:			30

tag: Type of Surgery:		Pneumectomy

tag: Location tumor:		Right middle lobe

tag: TNM stage:			T2N0M0

tag: Tumor stage:			IB

tag: Time to distant metastases as from surgery (months)	:	4

tag: Location of distant metastasis:				brain

tag: Dead/Alive:							dead

tag: Survival as from SCC resection (months):			6

tag: FEV1/FVC:			76 %

tag: Mix of DNA from 20 healthy women